Recruitment plan

The recruitment plan will be developed in conjunction with the recruitment centres and food manufacturers considering the capacity for appointments and food production, respectively. The rate of recruitment and randomization to the trial must match the rate of food production and delivery to the RC to ensure the availability of BEF for each participant.

3.2.1. Recruitment strategies

Accessing a target group with a specific health condition and motivating that group to take part in a dietary intervention trial can be challenging. If the health condition manifests through poor dietary and lifestyle choices it can be difficult to motivate such individuals to volunteer to a trial that involves changing firmly established behaviours. Experience from the PATHWAY- 27 study indicates that volunteers to a dietary intervention trial are often interested in their diet and health and therefore typically have a healthy metabolic profile. Consequently, they are not eligible to take part in a trial that requires participants with an unhealthy metabolic profile, for example elevated fasting blood triglycerides and/or low HDL cholesterol. Members of the public with an unhealthy metabolic profile, and who are often unaware of it, are less likely to be motivated to volunteer to take part in a dietary intervention trial.

Recruitment strategies are a key component to accessing the target group. Advertising the trial using the most effective media will help to improve the success rate of recruitment. The pilot study can be used to test different advertising methods and identify the most suitable for the target population. Advertising can be broadly focused to a non-specific audience, for example on a local radio station, or directly targeted to identify potential participants from a clinical patient register, for example at a local surgery.

A radio advert may reach many thousands of listeners and generate a lot of enquiries about the study. Translating those enquiries to participants requires an effective management system since it will not be feasible to speak directly with each advert respondent. A dedicated website or page on the RCs website is a useful first point of contact for enquiries where

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34 information about the trial can be conveyed. A link on the webpage can direct volunteers to an online survey that can be initially used to filter out individuals based on some of the exclusion criteria. For example, if treatment with statins is an exclusion criterion there can be a question on the survey that asks whether the respondent currently takes statins. If the provide an automatic message informing the individual that they are not eligible to participate. At the end of the survey, respondents who are eligible to continue with selection are asked to provide contact details that are collected by the RC. Subsequently potential participants can be contacted and asked to complete a secondary online survey that is used to collect more specific data from volunteers, such as date of birth, height and weight, physical activity, allergies, current medication and health conditions. This data can be used to identify potential participants and invite them to a screening appointment, and to exclude those who meet exclusion criteria.

3.2.2. Food distribution to participants

Distributing BEF to participants will be influenced by the BEF storage conditions, shelf-life and BEF with a long shelf-life, such as the PATHWAY- 27 milkshake powders that are stored ambiently and provided in a small sachet, pose very little problem for distribution. In this instance, it may only be necessary for one delivery. BEF that have a limited shelf-life may require more frequent deliveries. The bioactive stability of PATHWAY-27 biscuits dictated the shelf-life of the product. Short study duration, such as the 4-week pilot trial, only required one delivery to each participant. By contrast, a trial with duration longer than the shelf-life of the BEF would require more deliveries. BEF requiring cold storage are likely to pose the greatest storage problems. Firstly, it is important to note that the requirement for a freezer becomes an additional exclusion criterion. Providing that the participant has a freezer, the capacity needs to be considered. Short trial duration may be less problematic whereas a longer trial may necessitate multiple deliveries. In some instances, PATHWAY-27 pancakes had to be delivered to the home of the participants by research staff, since the participant could either not return home immediately after visiting the RC or they did not have suitable transport.

3.2.3. Participant retention and compliance

One of the most important aspects of conducting the trial is the retention and compliance of participants. Motivation for taking part in a trial varies between participants. Predominantly, in the PATHWAY-27 project, volunteers were motivated by potential for weight-loss, even though this was not a primary outcome of the study (this was clearly explained upon recruitment). If the participant does not feel that their personal objective for taking part in the trial is being met, they may lose motivation and withdraw.

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- Project funded under the Food, Agriculture and Fisheries, and Biotechnology theme (KBBE)

35 The ease with which a participant integrates the BEF into their usual diet will influence their

motivation. If the BEF are not well accepted then compliance to the study protocol will be impaired and may even lead to participants choosing to withdraw from the trial. Examples of how the foods could be consumed was provided (e.g. pancakes can be eaten on their own, or with sweet or savoury fillings). The time and effort that is required from a participant will also influence compliance and retention. A short trial that requires a participant to consume one BEF per day with relatively few questionnaires and visits to the RC will be more accepted than a longer trial with multiple foods and/or multiple questionnaires and visits to the RC.

Regular communication with each participant is essential to develop and maintain a good relationship. Participants who feel valued and well-informed will be more motivated to consume the BEF, complete the questionnaires and come to the RC when required. There are exceptions when participants are simply not well motivated, however in these cases it is unlikely that the research team will be able to improve compliance or prevent withdrawal from the trial.

3.2.4. Sample collection

For a human study investigating the effect of BEF on physiologically-relevant endpoints it is likely that biological samples will be required for analyses. The type of sample will depend on the selected endpoints but as a minimum requirement blood and possibly urine will be collected. It is common to collect a fasted sample from participants, particularly when measuring analytes that are influenced by consumption of food, such as glucose and triglycerides that will increase after a meal. To measure baseline values a fasting period of 10- 12 hours may be necessary.

In some cases, it might be sufficient to use a point-of-care device such as the Cardiochek Professional Analyser test system that can measure the concentration of single or multiple analytes, using analyte-specific test strips, in a small volume of capillary blood (15-40 µL) collected from a fingertip. This can be useful for screening volunteers as the results are available within a few minutes. In most cases it is more appropriate to collect larger volumes of blood to perform a wider range of analyses. In this case a trained phlebotomist will be required to perform venepuncture, typically on the antecubital fossa region.

It is essential to determine the biological markers prior to the start of the trial to ensure the most suitable blood collection tubes are used. The number and type of analytes to be measured will influence the tube volume and additive type. The volume of blood drawn should not be excessive to minimise discomfort to the participant. Ethically it will be important to justify the volume required. The correct tube type will depend on the analyte being measured and the analytical method. For example, to measure lipids it is necessary to collect blood into a tube containing a coagulant that causes the blood to clot. The tube is typically left for 30

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36 minutes at room temperature following blood collection to allow the clot to form. The tube

can then be centrifuged to separate the sample and extract serum. Multiple tube collections must adhere to recommendations for the order of draw to prevent potential cross- contamination of additives that may affect the accuracy of sample analyses. Current practice for the order of draw is:

1) Blood cultures 2) Citrate 3) Serum 4) Heparin 5) EDTA 6) Fluoride

Some analytical methods will require the sample to be refrigerated immediately after collection to prevent sample/analyte degradation, in other cases it may not be necessary. In all cases it is essential to know the correct storage conditions for the sample, use suitable storage containers that are appropriate for the storage conditions and to label the samples adequately to guarantee traceability.

3.2.5. Sample analysis

The protocols for sample analyses should be determined prior to the start of the trial. Changes to the analytical protocols, including changing equipment or laboratories, during the trial should be avoided as this could introduce a source of error into the results. Failure to manage the samples correctly may affect the accuracy of the analyses. If there are special conditions that are required for the processing and storage of samples they must be communicated to the research team involved with collection and processing of samples. For samples that are to be stored prior to being shipped to the analytical laboratory, it is essential that the storage conditions are maintained during transport, where necessary. Repeated freeze-thaw cycles may reduce the integrity of the sample and affect the results. For example, serum obtained for the analysis of lipids, glucose, thyroid, kidney and liver function in the PATHWAY-27 project, is stored at -80°C and must maintain its frozen state until it is required for analysis. By contrast, whole blood used to analyse single nucleotide polymorphisms (SNPs) is stored at - 20°C at the RC but can be shipped at ambient temperature.

Ideally all samples will be analysed by one laboratory to limit sources of random error. For multi monocentric studies, such as the PATHWAY-27 pilot studies, it is acceptable for sample analyses to be conducted at different laboratories however the analytical method, including the type of sample used for example serum or plasma, should be the same. For a multi- centre trial (PATHWAY-27 larger trial), the analyses should, where practicable, be done by a centralised laboratory. In some cases, it is not feasible to use one analytical laboratory due to

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37 the type of analysis that is to be performed. For example, a full blood count requires relatively fast analysis of whole blood to prevent artefacts from the EDTA anticoagulant that affect the results.

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- Project funded under the Food, Agriculture and Fisheries, and Biotechnology theme (KBBE)

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